Amphotericin B is the most widely used antifungal antibiotic for the treatment of life-threatening, deep-seated, systemic fungal infections. Although amphotericin B is the preferred antibiotic of choice for the systemic treatment of such life-threatening fungal infections, its use is limited by severe toxicity. Amphotericin B is presently produced by the cultivation of Streptomyces nodosus along with the toxic but substantially less active homolog, amphotericin A, as first described by Gold et al., 1956, Antibiotics Ann. 19955-19956:579-586. However, given the complexity of the synthetic pathway, should other strains of microorganisms be found to produce amphotericin B, it is expected that those new strains will produce amphotericin B by substantially the same pathway as is used by amphotericin B producing strains of Streptomyces.
Streptomyces nodosus is a streptomycete isolated from a soil sample taken from the Orinoco River region of Venezuela and identified as Streptomyces nodosus by Trejo et al., 1963, J. Bacteriol. 88:436-439. The microbial production and isolation of amphotericin B and amphotericin A from Streptomyces nodosus is described by Dutcher et al., U.S. Pat. Nos. 2,908,611 and 2,908,612, respectively, the disclosures of which are incorporated by reference herein in their entireties. Due to the toxic contribution of amphotericin A, which is a substantially less potent antifungal agent, amphotericin B for systemic use must have substantially all or most of the amphotericin A removed in order to achieve an acceptable therapeutic index. For example, amphotericin B for intravenous use must be at least 75% pure and contain no more than 5% amphotericin A (marketed as, e.g., FUNGIZONE, by E. R. Squibb & Sons, Inc.). The level of impurities in amphotericin B preparations depends upon the particular production strain, the cultivation conditions (electrolytes, pH and the like) and upon the purification methods applied to the crude fermentation product.
The structure and absolute configuration of amphotericin B was determined by X-ray crystallography and was reported in 1970 by Mechlinski et al., 1970, Tetrahedron Lett. 44:3873-3876., as shown below. ##STR1##
While the complete absolute configuration of amphotericin A has not been determined, it is very likely that the stereochemistry of amphotericin A is related, if not identical, to that of amphotericin B. ##STR2##
It is now believed that the only difference between these structurally homologous antibiotics is the reduction of the C.sub.28 -C.sub.29 double bond of the amphotericin B heptaene chromophore, resulting in the formation of the diene and tetraene structures in amphotericin A. Therefore, due to the close homology of these two compounds, the separation of amphotericin B from amphotericin A has heretofore proved to be a time-consuming and expensive chemical process that is often still based essentially upon the methods disclosed by U.S. Pat. Nos. 2,908,611 and 2,908,612, supra.
M. Shabas, et al, in Comparative Study of the Streptomycin Effect on Variation in Levorin and Amphotericin B Producers, Antibiotiki (Mosc.) 22(5), 1977, pp. 396-400, describes a study of the effect of streptomycin in the range of 1 to 6 .mu.g/ml. on the production of amphotericin B. However, the levels of streptomycin used in Shabas are cytotoxic, and would adversely affect the production of both amphotericin A and amphotericin B.
Thus, an improved method for the production of amphotericin B that provides substantially greater purity relative to its amphotericin A content has been long-sought and heretofore unavailable.